Method for enhancing immune response with peptide

ABSTRACT

An object of the present invention is to provide a safe and effective method for enhancing an immune response and a medicament for preventing or treating Alzheimer disease comprising amyloid β peptide that induces an enhanced immune response. An amyloid β peptide or a portion thereof with addition or insertion of cysteine and a method for enhancing an immune response using the peptide or a method for enhancing an immune response using the peptide together with an adjuvant. A medicament for preventing or treating Alzheimer disease comprising an amyloid β peptide or a portion thereof that induces an enhanced immune response. A DNA vaccine, that may have the same effect, comprising the gene encoding an amyloid β peptide or a portion thereof that induces an enhanced immune response with addition or insertion of cysteine.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/596,741, filed on Oct. 20, 2009, now U.S. Pat. No.8,106,015, which is a 35 U.S.C. §371 National Stage patent applicationof International patent application PCT/JP2008/057612, filed on Apr. 18,2008, which claims priority to Japanese patent applications JP2007-279083, filed on Oct. 26, 2007, and JP 2007-112060, filed on Apr.20, 2007.

TECHNICAL FIELD

The present invention relates to an immunogenic peptide that induces anenhanced immune response, comprising a peptide derived from amyloid β(Aβ), a causative substance of Alzheimer disease, or a portion thereofwith addition or insertion of cysteine. The present invention alsorelates to a medicament for preventing or treating Alzheimer diseasewhich comprises as an active ingredient said peptide. The immunogenicpeptide of the present invention, as sufficiently inducing an enhancedimmune response by itself without adjuvant, may safely be used as amedicament for the prevention or treatment without an adverse drugreaction associated with the use of adjuvant.

BACKGROUND ART

Immunity is one of self-protecting biological defense mechanisms againstinvading foreign organisms such as bacteria and viruses and includesinnate immunity associated with phagocytosis of leukocytes etc. andacquired immunity against a specific antigen/pathogen. A vaccine is amedicament for safely inducing the acquired immunity against a pathogenand recently has been used not only for protection but also fortreatment.

A vaccine includes a live vaccine, an inactivated vaccine, a componentvaccine, and the like. A live vaccine is highly immunogenic and has aprolonged immunological effect but also has a risk that pathogenicityremains or is reverted. An inactivated vaccine, prepared by treatingviruses with formalin etc. to remove the pathogenicity, is safer than alive vaccine but has a defect that the resulting immunity is notprolonged.

A component vaccine comprises as a main component an antigenic protein,prepared by extracting and purifying a protein having a vaccinal effectfrom a pathogen or artificially prepared by genetic engineeringtechniques or chemical procedures, and is highly safe free fromcontamination of unwanted proteins. Recently, a peptide vaccine as oneof component vaccines has been studied profoundly. A peptide refers to amolecule that consists of amino acids bound to each other throughpeptide linkage. Generally, a peptide with 10 or less amino acidresidues is called an oligopeptide, a peptide with less than 100 aminoacid residues is called a polypeptide (both an oligopeptide and apolypeptide are herein referred to as a “peptide”), and one with 100 ormore amino acid residues is called a protein.

Recently, amino acid sequences of various proteins and viral antigenicproteins have been determined. A peptide artificially synthesized basedon such an amino acid sequence is used as a vaccine, which is referredto as a peptide vaccine. The advantage of using a peptide as a vaccineis that a highly pure antigen can be obtained artificially withouthandling pathogenic microorganisms. On the other hand, it is difficultto obtain a sufficient immune effect since a peptide has a smallmolecular weight and hence is not recognized as a foreign substance inthe living body on immunization. Hence, a peptide is combined with alarge protein called a carrier protein so as to be recognized as aforeign substance and/or is administered together with an adjuvant(immunomodulator) in order to enhance immune effect. However, with thesetreatments, it is possible that an antibody against a carrier proteinmay be produced or an adverse side effect of an adjuvant mayunexpectedly be induced. Besides, while an adjuvant is proved to beeffective on research level, it is only an aluminum hydroxide gel thatis permitted in Japan for usage in human.

On the other hand, a peptide vaccine has been attempted for use forpreventing and/or treating diseases such as Alzheimer disease. Alzheimerdisease is one of dementia disorders and is associated with declinedcognitive function and change in personality as principal symptoms. Theincreasing number of patients along with rapid increase of agingpopulation has become a social issue. Alzheimer disease's pathologicalindications include three features of atrophy and/or fall-off ofneurons, formation of senile plaques due to aggregation and/ordeposition of Aβ and neurofibrillary changes due to abnormal tauproteins. Onset of Alzheimer disease is initiated by deposition of Aβpeptides (senile plaque formation) followed by denaturing and fall-ofneurons with increase in Aβ deposition. The deposition of Aβ peptidesthen trigger deposition of tau proteins followed by neurofibrillarychanges. Aβ peptide is derived from an amyloid peptide precursor (APP)existing in the brain and the body. In normal process, APP is cleaved byα-secretase in the middle and then by γ-secretase in the C-terminal togenerate a P3 peptide which is subsequently degraded completely. In thecase of Aβ peptide deposition, APP is cleaved by β-secretase and then byγ-secretase in the C-terminal to generate Aβ peptides consisting of 40or 42 amino acid resides (Aβ40, Aβ42). Among these, Aβ42, easilyaggregated and deposited, is extracellularly secreted to beinsolubilized, and aggregated and deposited to form senile plaques.Increase in production and accumulation of Aβ42 peptides would affect asynapse. Further, microglial cells and astrocytes are gathered aroundthe aggregated Aβ peptides. It is thought that damages in the synapseand the neurite further progress to lead to degeneration and cell deathof neurons, resulting in dementia.

Nowadays, targeting Aβ peptides, a method of treatment is considered fordecreasing Aβ peptides, including, for instance, inhibition of theaction of secretases which produce Aβ peptides, use of an Aβ degradingenzyme which may degrade the produced Aβ peptides, use of a vaccine oran antibody for removing those extracellularly excreted and thoseaggregated, and the like.

Approach of treating Alzheimer disease with a vaccine was first reportedby Schenk et al. (Non-patent reference 1), which comprises administeringAβ42 peptides together with an adjuvant by intramuscular injection tothereby produce an antibody against Aβ to remove the accumulated Aβpeptides. A clinical trial for the vaccine was performed byadministering intramuscularly a medicament comprising the Aβ42 peptidetogether with a purified saponin as an adjuvant. As a result, it wasshown that an antibody specific to Aβ peptide was produced in Alzheimerdisease patients by administration of the vaccine and that theproduction of the antibody specific to Aβ peptide could retard thedevelopment of cognitive disturbance in Alzheimer disease patients(Non-patent reference 2) and it was proved that senile plaques weredisappeared (Non-patent reference 3). However, since seriousmeningoencephalitis was observed in some subjects, the clinical trialwas discontinued. It is supposed that one of causes of the adverse sideeffect is the adjuvant contained in the vaccine. Accordingly, for apeptide vaccine, development of a formulation that is efficient and safeis strongly desired.

-   Non-patent reference 1: Schenk D, Barbour R, Dunn W, Gordon G,    Grajeda H, Guido T, et al., Immunization with amyloid-beta    attenuates Alzheimer disease -disease-like pathology in the PDAPP    mouse. Nature 1999; 400: p. 173-177-   Non-patent reference 2: Fox N C, Black R S, Gilman S, Rossor M N,    Griffith S G, Jenkins L, et al. Effects of A beta immunization    (AN1792) on MRI measures of cerebral volume in Alzheimer disease.    Neurology 2005; 64: p. 1563-1572-   Non-patent reference 3: Nicoll J A, Wilkinson D, Holmes C, Steart P,    Markham H, Weller R O. Neuropathology of human Alzheimer disease    after immunization with amyloid-beta peptide: a case report. Nat Med    2003; 9: p. 448-452

DISCLOSURE OF THE INVENTION Technical Problem to be Solved by theInvention

As mentioned above, there is a concern that the substances known to havean adjuvant effect may exert adverse side effects contrary to theadjuvant effect. Thus, an object of the present invention is to providea novel method for enhancing an immune response using a substance whichhas already been confirmed to be safe for the living body and animmunogenic peptide that induces an enhanced immune response for use insaid method.

Means for Solving the Problems

The present inventors have earnestly investigated a method forimmunization and for enhancing immunization that is safe for the livingbody, efficacious and inexpensive, and as a result, have found that acapacity of inducing an enhanced immune response of a peptide ofinterest may be enhanced by addition or insertion of a cysteine residue,an amino acid constituting a naturally-occurring protein, to therebyaccomplish the present invention.

The present invention relates to a method of enhancing an immuneresponse inducing property of a peptide derived from Aβ, which is acausative substance of Alzheimer disease, or a portion thereof,characterized by addition or insertion of cysteine to Aβ peptide or aportion thereof. The present invention includes the followings:

(1) An immunogenic peptide that induces an enhanced immune responsecomprising an amyloid β peptide or a portion thereof with addition orinsertion of cysteine or with addition of a peptide containing cysteine;

(2) The immunogenic peptide according to the above (1) wherein cysteineis added at the N-terminal or the C-terminal or both of the amyloid βpeptide or a portion thereof;

(3) The immunogenic peptide according to the above (1) or (2) whereincysteine is added at the C-terminal of the amyloid β peptide or aportion thereof;

(4) The immunogenic peptide according to any of the above (1) to (3)wherein 1 or 2 molecules of cysteine are added;

(5) The immunogenic peptide according to the above (1) wherein thepeptide containing cysteine is added at the C-terminal of the amyloid βpeptide or a portion thereof;

(6) The immunogenic peptide according to the above (1) wherein cysteineis inserted into the amyloid β peptide or a portion thereof;

(7) The immunogenic peptide according to the above (6) wherein cysteineis inserted between the 18th and 19th amino acid residues, between the25th and 26th amino acid residues, or between the 28th and 29th aminoacid residues counted from the N-terminus of an amyloid β peptide or aportion thereof;

(8) The immunogenic peptide according to any of the above (1) to (7)wherein the amyloid β peptide or a portion thereof consists of the aminoacid sequence of SEQ ID NO: 34 or a portion thereof;

(9) The immunogenic peptide according to any of the above (1) to (8)which consists of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ IDNO: 52, SEQ ID NO: 54 and SEQ ID NO: 56;

(10) A medicament for preventing or treating Alzheimer disease whichcomprises as an active ingredient the immunogenic peptide of any of theabove (1) to (9);

(11) A medicament for preventing or treating Alzheimer disease whichcomprises as an active ingredient the immunogenic peptide of any of theabove (1) to (9) together with an adjuvant;

(12) A DNA vaccine effective for preventing or treating Alzheimerdisease which comprises a gene fragment encoding the amino acid sequenceof the immunogenic peptide of any of the above (1) to (9);

(13) A method for enhancing an immune response which comprises using animmunogenic peptide that induces an enhanced immune response comprisingan amyloid β peptide or a portion thereof with addition or insertion ofcysteine or with addition of a peptide containing cysteine;

(14) A method for enhancing an immune response which comprises using animmunogenic peptide that induces an enhanced immune response comprisingan amyloid β peptide or a portion thereof with addition or insertion ofcysteine or with addition of a peptide containing cysteine together withan adjuvant;

(15) A method for enhancing an immune response which comprises using asa DNA vaccine a vector containing a gene fragment encoding an amino acidsequence of an immunogenic peptide that induces an enhanced immuneresponse comprising an amyloid β peptide or a portion thereof withaddition or insertion of cysteine or with addition of a peptidecontaining cysteine.

Effects of the Invention

The present invention provides an immunogenic peptide that induces anenhanced and sufficient immune response even if it is used alone withoutan adjuvant. According to the present invention, merely by addition orinsertion of cysteine residues to the amyloid β peptide or a portionthereof, antibody production of an immunogenic peptide may be enhanced.Therefore, there is no disadvantage associated with the use of anadjuvant to allow for easier design of a drug formulation.

The immunogenic peptide of the present invention that induces anenhanced immune response, when administered to the living body, mayrapidly and abundantly induce an antibody specific to the peptide inblood. No toxicity of cysteine is known but rather cysteine and itsrelated substances are known to have an antitoxic effect in the livingbody and therefore the immunogenic peptide of the present invention thatinduces an enhanced immune response may be used in the body very safely.

The immunogenic peptide of the present invention that induces anenhanced immune response, when used as a peptide vaccine, may be thesimplest vaccine comprising as an active ingredient only the immunogenicpeptide with addition or insertion of cysteine. Such immunogenic peptidewith addition or insertion of cysteine may be prepared by chemicalsynthesis without biological synthesis and hence in higher uniformitythan the conventional component vaccines. Additionally, with the lowestrisk of toxicity, infection and decrease in quality due tocontamination, a safer vaccine may be provided.

A peptide preparation comprising the immunogenic peptide of the presentinvention that induces an enhanced immune response may be administerednot only by injection such as subcutaneous or intramuscularadministration but also by oral, transnasal or transdermaladministration, which would avoid stress and medical accidents caused bysyringe needle.

BEST MODE FOR CARRYING OUT THE INVENTION

For preparing the immunogenic peptide of the present invention thatinduces an enhanced immune response, a cysteine residue may be added orinserted to the Aβ peptide or a portion thereof or alternatively anucleotide sequence encoding cysteine may be added or inserted to theDNA or RNA sequence of the Aβ peptide or a portion thereof forexpression. With respect to the position of addition or insertion ofcysteine in the Aβ peptide or a portion thereof, in the case ofaddition, cysteine may be added at the N-terminal or the C-terminal orboth of the peptide and, in the case of insertion, cysteine may beinserted at any position in the peptide. Addition at the C-terminal ofthe peptide is preferable wherein 1 or 2 cysteine residue(s) may beadded. However, insofar as the immune response enhancing effect may beobtained, the position of addition and insertion or the number ofcysteine residues are not especially limited. In another aspect of thepresent invention, a peptide containing cysteine, in place of cysteine,may also be added at the C-terminal of the peptide.

The Aβ peptide consists of 42 amino acid residues (Aβ42) and has thefollowing amino acid sequence:

(SEQ ID NO: 34) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA.Cysteine (Cys) may be added or inserted to the Aβ peptide or a portionthereof to provide the immunogenic peptide of the present invention thatinduce an enhanced immune response. A portion of the Aβ peptide includesthose consisting of an amino acid sequence comprising at least the1st-28th of the aforementioned amino acid sequence of Aβ42.Alternatively, a peptide containing cysteine may be added to the Aβpeptide or a portion thereof. The thus obtained immunogenic peptide thatinduces an enhanced immune response is efficacious for preventing ortreating Alzheimer disease.

Specifically, a preferred example of the immunogenic peptide of thepresent invention that induces an enhanced immune response includes thefollowing Aβ peptides with addition or insertion of cysteine wherein anadded or inserted cysteine residue is underlined.

-   (1) Peptide with addition of 1 molecule at the C-terminal:

28AACys: (SEQ ID NO: 6) DAEFRHDSGYEVHHQKLVFFAEDVGSNKC 29AACys:(SEQ ID NO: 8) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGC 30AACys: (SEQ ID NO: 10)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAC 31AACys: (SEQ ID NO: 12)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIC 35AACys: (SEQ ID NO: 21)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMC 36AACys: (SEQ ID NO: 23)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVC 37AACys: (SEQ ID NO: 25)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGC 38AACys: (SEQ ID NO: 27)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGC 39AACys: (SEQ ID NO: 29)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVC 40AACys: (SEQ ID NO: 32)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVC 42AACys: (SEQ ID NO: 36)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAC

-   (2) Peptide with addition of 2 molecules at the C-terminal:

28AACysCys: (SEQ ID NO: 38) DAEFRHDSGYEVHHQKLVFFAEDVGSNKCC 

-   (3) Peptide with addition of 1 molecule at the N-terminal:

Cys28AA: CDAEFRHDSGYEVHHQKLVFFAEDVGSNK (SEQ ID NO: 40)

-   (4) Peptide with addition of each 1 molecule at the C-terminal and    the N-terminal:

Cys28AACys: CDAEFRHDSGYEVHHQKLVFFAEDVGSNKC (SEQ ID NO: 42)

-   (5) Peptide with insertion of 1 molecule:

28AA18Cys: (SEQ ID NO: 46) DAEFRHDSGYEVHHQKLVCFFAEDVGSNK 28AA25Cys:(SEQ ID NO: 48) DAEFRHDSGYEVHHQKLVFFAEDVGCSNK 33AA28Cys: (SEQ ID NO: 50)DAEFRHDSGYEVHHQKLVFFAEDVGSNKCGAIIG 35AA28Cys: (SEQ ID NO: 52)DAEFRHDSGYEVHHQKLVFFAEDVGSNKCGAIIGLM

-   (6) Peptide with addition of 1 molecule at the C-terminal+addition    of an exogenous peptide (the exogenous peptide is positioned at the    C-terminal of the added cysteine residue):

28AACysTTD: (SEQ ID NO: 54) DAEFRHDSGYEVHHQKLVFFAEDVGSNKCTTD28AACysEIFEFTTD: (SEQ ID NO: 56) DAEFRHDSGYEVHHQKLVFFAEDVGSNKCEIFEFTTD

Among the Aβ peptides with addition or insertion of cysteineaforementioned, 28-amino acid Aβ peptide with addition of cysteine(28AACys: SEQ ID NO: 6), 29-amino acid Aβ peptide with addition ofcysteine (29AACys: SEQ ID NO: 8), 31-amino acid Aβ peptide with additionof cysteine (31AACys: SEQ ID NO: 12), 35-amino acid Aβ peptide withaddition of cysteine (35AACys: SEQ ID NO: 21), 36-amino acid Aβ peptidewith addition of cysteine (36AACys: SEQ ID NO: 23), 39-amino acid Aβpeptide with addition of cysteine (39AACys: SEQ ID NO: 29), 40-aminoacid Aβ peptide with addition of cysteine (40AACys: SEQ ID NO: 32),42-amino acid Aβ peptide with addition of cysteine (42AACys: SEQ ID NO:36), 28-amino acid Aβ peptide with addition of 2 cysteines at theC-terminal (28AACysCys: SEQ ID NO: 38) and 28-amino acid Aβ peptide withinsertion of cysteine between 18th-19th amino acid residues (28AA18Cys:SEQ ID NO: 46) could particularly induce the enhanced immune responseand thus may be used to prevent or treat Alzheimer diseaseefficaciously.

According to the present invention, a peptide that induces an enhancedimmune response may be prepared by adding cysteine at the N-terminal,the C-terminal, or both of the N-terminal and the C-terminal of the Aβpeptide or a portion thereof or by inserting cysteine into said peptide.Whether a peptide obtained after addition or insertion of cysteineexerts an immune response-enhancing effect may be corroborated byimmunization of mice with the peptide using the conventional techniquesand determining an anti-Aβ IgG antibody titer in blood. Thus, thepresent invention also provides a method of preparing an immunogenicpeptide that induces an enhanced immune response characterized by addingone or more cysteines at the N-terminal, the C-terminal, or both at theN-terminal and the C-terminal of the Aβ peptide or a portion thereof orinserting one or more cysteines to the peptide, immunizing an animalwith the resulting peptide, and then determining an anti-Aβ IgG antibodytiter in blood of the animal.

A peptide preparation containing the immunogenic peptide of the presentinvention that has an enhanced immune response-inducing property may beadministered by any route of administration such as subcutaneous,transdermal, intramuscular, oral, or transnasal.

While the immunogenic peptide of the present invention that induces anenhanced immune response may provide sufficient immunization even if itis administered alone without an adjuvant, it may provide furthersufficient immunization if in combination with an adjuvant. It will thusbecome possible to select various types of adjuvants such as e.g. anadjuvant attached importance to an effect of enhancing immunization oran adjuvant attached importance to a safety.

Moreover, a vector which comprises a gene fragment encoding each of theimmunogenic peptides of the present invention as listed above thatinduce an enhanced immune response obtained by addition or insertion ofcysteine to the Aβ peptide or a portion thereof may be used as a DNAvaccine for efficaciously preventing and treating Alzheimer disease. Anucleotide sequence encoding cysteine includes e.g. tgt but may be anysequence as far as it encodes cysteine. A gene fragment encoding theamino acid sequence of the Aβ peptide (Aβ42) consisting of the 42 aminoacid residues mentioned above (SEQ ID NO: 34) is described below.However, the nucleotide sequence described below represents a typicalgene sequence of the Aβ peptide but any gene sequence may be employedinsofar as it encodes the same amino acid sequence.

gatgcagaat tccgacatga ctcaggatat gaagttcatc atcaaaaatt ggtgttctttgcagaagatg tgggttcaaa caaaggtgca atcattggac tcatggtggg cggtgttgtc atagcg(SEQ ID NO: 35)

An example of a gene fragment encoding each of the immunogenic peptidesof the present invention as listed above that induce an enhanced immuneresponse obtained by addition or insertion of cysteine to the Aβ peptideor a portion thereof includes those described below. However, thenucleotide sequences described below represent a typical gene sequenceencoding each of the peptides mentioned above but any gene sequence maybe employed insofar as it encodes the same amino acid sequence.

Gene fragment encoding 26AACys: (SEQ ID NO: 2)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcatgtGene fragment encoding 27AACys: (SEQ ID NO: 4)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa ctgtGene fragment encoding 28AACys: (SEQ ID NO: 7)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaatgtGene fragment encoding 29AACys: (SEQ ID NO: 9)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaaggttgtGene fragment encoding 30AACys: (SEQ ID NO: 11)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaaggtgca tgtGene fragment encoding 31AACys: (SEQ ID NO: 13)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaaggtgca atctgtGene fragment encoding 32AACys: (SEQ ID NO: 15)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaaggtgca atcatttgtGene fragment encoding 33AACys: (SEQ ID NO: 17)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaaggtgca atcattggat gtGene fragment encoding 34AACys: (SEQ ID NO: 19)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaaggtgca atcattggac tctgtGene fragment encoding 35AACys: (SEQ ID NO: 22)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaaggtgca atcattggac tcatgtgt Gene fragment encoding 36AACys:(SEQ ID NO: 24) gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaaggtgca atcattggac tcatggtgtg t Gene fragment encoding 37AACys:(SEQ ID NO: 26) gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaaggtgca atcattggac tcatggtggg ctgt Gene fragment encoding 38AACys:(SEQ ID NO: 28) gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaaggtgca atcattggac tcatggtggg cggttgt Gene fragment encoding 39AACys:(SEQ ID NO: 30) gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaaggtgca atcattggac tcatggtggg cggtgtttgtGene fragment encoding 40AACys: (SEQ ID NO: 33)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaaggtgca atcattggac tcatggtggg cggtgttgtc tgtGene fragment encoding 42AACys: (SEQ ID NO: 37)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaaggtgca atcattggac tcatggtggg cggtgttgtc atagcgtgtGene fragment encoding 28AACysCys: (SEQ ID NO: 39)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaatgttgtGene fragment encoding Cys28AA: (SEQ ID NO: 41)tgtgatgcag aattccgaca tgactcagga tatgaagttcatcatcaaaa attggtgttc tttgcagaag atgtgggttc aaacaaaGene fragment encoding Cys28AACys: (SEQ ID NO: 43)tgtgatgcag aattccgaca tgactcaggatat gaagttcatcatcaaaa attggtgttc tttgcagaag atgtgggttc aaacaaatgtGene fragment encoding 28AA18Cys: (SEQ ID NO: 47)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgtgtttc tttgcagaag atgtgggttc aaacaaa Gene fragment encoding 28AA25Cys: (SEQ ID NO: 49)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttgttcaaa caaaGene fragment encoding 33AA28Cys: (SEQ ID NO: 51)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaatgtggt gcaatcattg gaGene fragment encoding 35AA28Cys: (SEQ ID NO: 53)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaatgtggt gcaatcattg gactcatg Gene fragment encoding 28AACysTTD(SEQ ID NO: 54): (SEQ ID NO: 55)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaa caaagact actgacGene fragment encoding 28AACysEIFEFTTD (SEQ ID NO: 56): (SEQ ID NO: 57)gatgcagaat tccgacatga ctcaggatat gaagttcatcatcaaaaatt ggtgttcttt gcagaagatg tgggttcaaacaaatgtgaa atcttcgaat tcactactgac

The present invention is explained in more detail by means of thefollowing Examples but should not be construed to be limited thereto.

EXAMPLE 1

Comparison of Antibody Inducing Ability Between Aβ Peptides with andwithout Addition of Cysteine

(1) Preparation of Aβ Peptides with Addition of Cysteine

28AA: (SEQ ID NO: 5) DAEFRHDSGYEVHHQKLVFFAEDVGSNK 28AACys:(SEQ ID NO: 6) DAEFRHDSGYEVHHQKLVFFAEDVGSNKC 35AA: (SEQ ID NO: 20)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM 35AACys: (SEQ ID NO: 21)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMC

The above peptides were synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution. To 100 μL ofthe stock solution was added 900 μL of saline to 0.5 mg/mL of theconcentration and the mixture was dispensed into 1.5 mL tube (immunogen)and stored at −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 16 mice were divided into 4 groups each comprising 4 mice: Group 1administered with 28AA; Group 2 administered with 28AACys; Group 3administered with 35AA; and Group 4 administered with 35AACys.

(4) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(5) Blood Sampling

On Day 7 of the 2nd immunization, 50 to 150 μL of blood was collectedfrom the tail vein. Further, on Day 7 from the final 3rd immunization,blood was collected from the abdominal aorta of all mice underanesthesia with pentobarbital sodium (Kyoritsu Seiyaku Corporation,Somnopentyl). The sampling blood was transferred to the Microtainer(Becton Dickinson Co., Ltd), adequate clotting has occurred at roomtemperature and then centrifuged at 5,000 rpm for 10 min. Each of theseparated serum was dispensed into two 0.5 mL tubes and stored at −80°C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

The Aβ peptide (1-40 amino acid sequence:

(SEQ ID NO: 31) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVsynthesized by Hokkaido System Science Co., Ltd.), diluted to 10 μg/mLwith 0.1M carbonate buffer, pH9.6, was added to 8-well strips (NalgeNunc K.K., Immobilizer Amino) at 100 μL/well and left to incubate at 4°C. overnight for immobilization. On the following day, each well waswashed 3 times with 300 μL of PBS containing 0.05% Tween20 (PEST), addedwith 10 mM ethanolamine at 300 μL/well and left to incubate at roomtemperature for 1 hour.

After 1 hour, the 10 mM ethanolamine was fully removed and a specimendiluted with PEST by 50- to 10000-fold was added to each well at 100μL/well. After reaction at room temperature for 1 hour, the dilutedserum added was discarded and each well was washed 3 times with 300μL/well of PEST. After washing, the wash solution in the well was fullyremoved, an HRP-labeled anti-mouse IgG goat antibody (American Curlex,A131PS) diluted with the solution for the specimen dilution by 2000-foldwas added at 100 μL/well followed by reaction at room temperature for 1hour. After the reaction, the solution for labeled antibody dilution wasdiscarded and each well was washed twice with 300 μL/well of PBST andtwice with the equivalent amount of distilled water, to which 100μL/well of a chromogenic substrate solution TMB+ (Dako Inc.) was addedfollowed by reaction at room temperature for 30 min. under shading.Then, 100 μL/well of 1N sulfuric acid was added to quench developmentand optical density at 450 nm (OD450 value) was measured.

A commercially available monoclonal antibody to Aβ (CHEMI-CONCorporation, MAB1560) was used as standard serum. The standard serum wasdiluted with PBST to 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 ng/mL toprepare standards for the antibody titer measurement. An anti-Aβ IgGantibody of each murine serum specimen was determined and simultaneouslythe OD450 value of each diluted specimen was measured. An anti-Aβ IgGantibody titer of each murine serum specimen was calculated using theunit of the resulting standards and the standard curve of the OD450value.

Table 1 shows the calculated anti-Aβ antibody titer of the murine serumin each of the immunization groups. As shown in Table 1, as compared tothe immunization with Aβ peptide fragments without addition of cysteine(28AA, 35AA), the immunization with Aβ peptide fragments with additionof cysteine (28AACys, 35AACys) provided a higher antibody titer againstAβ. Increase in the antibody titer was observed for the 28AA withaddition of cysteine by about 39-fold and for the 35AA with addition ofcysteine by about 26-fold.

TABLE 1 Example 2 comparison of antibody inducing ability between Aβpeptide with addition of cysteine and carrier- linked Aβpeptide (with/without adjuvant) (1) Preparation of Aβpeptide with addition of cysteine and carrier-linked Aβ peptide 28AACys:DAEFRHDSGYEVHHQKLVFFAEDVGSNKC (SEQ ID NO: 6) 28AA-KLH:DAEFRHDSGYEVHHQKLVFFAEDVGSNK-C-KLH (SEQ ID NO: 58)

The above peptides were synthesized (KITAYAMA LABES Co., Ltd.) anddiluted with saline to obtain a 1 mg/mL stock solution which was storedat −80° C. or lower till use. KLH (keyhole limpet hemocyanin) is acarrier protein of 60 kDa. Cys in the 28AA-KLH was used as a linker tocombine the 28AA peptide with KLH and was not intended to enhance theimmune response effect. To combine a carrier protein with theimmunogenic peptide using Cys is a routine method for peptideimmunization.

(2) Adjuvant

Freund's complete adjuvant (hereafter referred to as “FCA”), Freund'sincomplete adjuvant (hereafter referred to as “FICA”), (GERBU, #1841,#1842), Gerbu adjuvant MM (GERBU, #3001.0106) and Alhydrogel ‘85’ 2%(Brenntag) which were commercially available were used as adjuvants.

(3) Administered Mice

Male C57BL/6 mice (9 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(4) Immunization Groups

The 25 mice were divided into 5 groups each comprising 5 mice: Group 1administered with 28AACys; Group administered with 28AA-KLH; Group 3administered with 28AA-KLH+FCA/FICA; Group 4 administered with28AA-KLH+Gerbu adjuvant MM; and Group 5 administered with28AA-KLH+Alhydrogel ‘85’ 2%.

(5) Dose and Preparation of Sample for Administration

A primary dose of Aβ peptide was 100 μg per animal, and second and thirddoses were 50 μg per animal. Namely, to prepare the primary dose for thegroup administered with 28AACys, 0.5 mL of 28AACys and 0.5 mL of salinewere mixed, and to prepare the second and third doses, 0.25 mL of28AACys and 0.75 mL of saline were mixed. To prepare the primary dosefor the group administered with 28AA-KLH, 0.5 mL of 28AA-KLH and 0.5 mLof saline were mixed, and to prepare the second and third doses, 0.25 mLof 28AA-KLH and 0.75 mL of saline were mixed. To prepare the primarydose for the group administered with 28AA-KLH+FCA/FICA, 0.5 mL of28AA-KLH and 0.5 mL of FCA were mixed to be emulsified, and to preparethe second and third doses, 0.25 mL of 28AA-KLH, 0.25 mL of saline and0.5 mL of FICA were mixed to be emulsified. To prepare the primary dosefor the group administered with 28AA-KLH+Gerbu adjuvant MM, 0.5 mL of28AA-KLH, 0.4 mL of saline and 0.1 mL of Gerbu adjuvant MM were mixed,and to prepare the second and third doses, 0.25 mL of 28AA-KLH, 0.65 mLof saline and 0.1 mL of Gerbu adjuvant MM were mixed. To prepare thefirst dosage for the group administered with 28AA-KLH+Alhydrogel ‘85’2%, 0.5 mL of 28AA-KLH and 0.5 mL of Alhydrogel ‘85’ 2% were mixed, andto prepare the second and third doses, 0.25 mL of 28AA-KLH, 0.25 mL ofsaline and 0.5 mL of Alhydrogel ‘85’ 2% were mixed.

(6) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(7) Blood Sampling

On Day 7 from the final 3rd immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(8) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 2 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization groups. As shown in Table 2, the Aβpeptide fragment with addition of a carrier KLH together with Freund'scomplete/incomplete adjuvant provided the highest antibody titer againstAβ. The secondly highest antibody titer was provided by the Aβ peptidefragment with addition of cysteine, which was significantly higher thanthose of using as a adjuvant Gerbu adjuvant MM (GERBU) or Alhydrogel‘85’ 2% or of the Aβ peptide fragment with addition of a carrier KLH.

TABLE 2 Example 3 Comparison of antibody inducing ability among Aβpeptides with addition of cysteine having26-40 amino acid sequence in length (1) Preparation of Aβpeptides with addition of cysteine 26AACys: (SEQ ID NO: 1)DAEFRHDSGYEVHHQKLVFFAEDVGSC 27AACys: (SEQ ID NO: 3)DAEFRHDSGYEVHHQKLVFFAEDVGSNC 28AACys: (SEQ ID NO: 6)DAEFRHDSGYEVHHQKLVFFAEDVGSNKC 29AACys: (SEQ ID NO: 8)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGC 30AACys: (SEQ ID NO: 10)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAC 31AACys: (SEQ ID NO: 12)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIC 32AACys: (SEQ ID NO: 14)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIC 33AACys: (SEQ ID NO: 16)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGC 34AACys: (SEQ ID NO: 18)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLC 35AACys: (SEQ ID NO: 21)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMC 36AACys: (SEQ ID NO: 23)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVC 37AACys: (SEQ ID NO: 25)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGC 38AACys: (SEQ ID NO: 27)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGC 39AACys: (SEQ ID NO: 29)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVC 40AACys: (SEQ ID NO: 32)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVC

The above peptides were synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution. To 100 μL ofthe stock solution was added 900 μL of saline to 0.5 mg/mL of theconcentration and the mixture was dispensed into 1.5 mL tube (immunogen)and stored at −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 60 mice were divided into 15 groups each comprising 4 mice: Group 1administered with 26AACys; Group administered with 27AACys; Group 3administered with 28AACys; Group 4 administered with 29AACys; Group 5administered with 30AACys; Group 6 administered with 31AACys; Group 7administered with 32AACys; Group 8 administered with 33AACys; Group 9administered with 34AACys; Group 10 administered with 35AACys; Group 11administered with 36AACys; Group 12 administered with 37AACys; Group 13administered with 38AACys; Group 14 administered with 39AACys; and Group15 administered with 40AACys.

(4) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(5) Blood Sampling

On Day 7 from the final 3rd immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 3 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization groups. As shown in Table 3, it wasobserved that, among the groups administered with Aβ peptides withaddition of cysteine which have 26 to 40 amino acid residues in length,those with 28 or more amino acid residues in length had the antibodyinducing ability against Aβ. In particular, 28AACys, 29AACys, 31AACys,35AACys, 36AACys, 39AACys, and 40AACys showed the higher antibody titer.

TABLE 3 Example 4 Comparison of antibody inducing ability among Aβpeptides without addition of cysteine, withaddition of 1 molecule of cysteine and withaddition of 2 molecules of cysteine (1) Preparation of Aβpeptides without and with addition of cysteine. 28AA:DAEFRHDSGYEVHHQKLVFFAEDVGSNK (SEQ ID NO: 5) 28AACys:DAEFRHDSGYEVHHQKLVFFAEDVGSNKC (SEQ ID NO: 6) 28AACysCys:DAEFRHDSGYEVHHQKLVFFAEDVGSNKCC (SEQ ID NO: 38)

The above peptides were synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution, which wasstored at −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 12 mice were divided into 3 groups each comprising 4 mice: Group 1administered with 28AA; Group 2 administered with 28AACys; and Group 3administered with 28AACysCys.

(4) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(5) Blood Sampling

On Day 7 from the final 4th immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 4 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization Groups. As shown in Table 4, almostthe same antibody inducing ability was found in the groups administeredwith 28AACys or 28AACysCys.

TABLE 4 Example 5 Comparison of antibody inducing ability among Aβpeptides with addition of cysteine at the C-terminal, the N-terminal and both terminals (1) Preparation of Aβpeptides with addition of cysteine 28AACys:DAEFRHDSGYEVHHQKLVFFAEDVGSNKC (SEQ ID NO: 6) Cys28AA:CDAEFRHDSGYEVHHQKLVFFAEDVGSNK (SEQ ID NO: 40) Cys28AACys:CDAEFRHDSGYEVHHQKLVFFAEDVGSNKC (SEQ ID NO: 42)

The above peptides were synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution, which wasstored at −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 12 mice were divided into 3 groups each comprising 4 mice: Group 1administered with 28AACys; Group 2 administered with Cys28AA; and Group3 administered with Cys28AACys.

(4) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T26135) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(5) Blood Sampling

On Day 7 from the final 4th immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl) and the mice weresacrificed. The sampling blood was transferred to the Microtainer(Becton Dickinson Co., Ltd), adequate clotting has occurred at roomtemperature and then centrifuged at 5,000 rpm for 10 min. Each of theseparated serum was dispensed into two 0.5 mL tubes and stored at −80°C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 5 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization groups. As shown in Table 5,regardless of position of Cys addition, the antibody inducing abilitywas found in all the groups.

TABLE 5 Example 6 Comparison of antibody inducing ability among Aβpeptide sequences with insertion of cysteine (1) Preparation of Aβpeptides with insertion of cysteine 28AA7Cys: (SEQ ID NO: 44)DAEFRHDCSGYEVHHQKLVFFAEDVGSNK 28AA10Cys: (SEQ ID NO: 45)DAEFRHDSGYCEVHHQKLVFFAEDVGSNK 28AA18Cys: (SEQ ID NO: 46)DAEFRHDSGYEVHHQKLVCFFAEDVGSNK 28AA25Cys: (SEQ ID NO: 48)DAEFRHDSGYEVHHQKLVFFAEDVGCSNK 33AA28Cys: (SEQ ID NO: 50)DAEFRHDSGYEVHHQKLVFFAEDVGSNKCGAIIG 35AA28Cys: (SEQ ID NO: 52)DAEFRHDSGYEVHHQKLVFFAEDVGSNKCGAIIGLM 28AACys: (SEQ ID NO: 6)DAEFRHDSGYEVHHQKLVFFAEDVGSNKC

The above peptides were synthesized (Sigma-Aldrich Japan K.K.) anddiluted with saline to obtain a 5 mg/mL stock solution, which was storedat −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 28 mice were divided into 7 groups each comprising 4 mice: Group 1administered with 28AA7Cys; Group 2 administered with 28AA10Cys; Group 3administered with 28AA18Cys; Group 4 administered with 28AA25Cys; Groupadministered with 33AA28Cys; Group 6 administered with 35AA28Cys; andGroup 7 administered with 28AACys.

(4) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(5) Blood Sampling

On Day 7 from the final 4th immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 6 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization Groups. As shown in Table 6, theantibody inducing ability was found in the groups administered with28AA18Cys, 28AA25Cys, 33AA28Cys, 35AA28Cys or 28AACys. In particular, ahigher antibody inducing ability was found for the group administeredwith 28AA18Cys, the 28 amino acids Aβ peptide with insertion of cysteinebetween the 18th and 19th amino acid residue, as compared to the groupadministered with 28AACys, the 28 amino acid Aβ peptide with addition ofcysteine at the C-terminal.

TABLE 6 Example 7 Assessment of antibody inducing ability of Aβpeptide with addition of cysteine together withaddition of exogenous amino acid sequence (not derived from Aβ peptide)(1) Preparation of Aβ peptides with and without addition of cysteine28AA: (SEQ ID NO: 5) DAEFRHDSGYEVHHQKLVFFAEDVGSNK 28AACys:(SEQ ID NO: 6) DAEFRHDSGYEVHHQKLVFFAEDVGSNKC 28AACysTTD: (SEQ ID NO: 54)DAEFRHDSGYEVHHQKLVFFAEDVGSNKCTTD 28AACysEIFEFTTD: (SEQ ID NO: 56)DAEFRHDSGYEVHHQKLVFFAEDVGSNKCEIFEFTTD

The above peptides were synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution, which wasstored at −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 16 mice were divided into 4 groups each comprising 4 mice: Group 1administered with 28AA; Group 2 administered with 28AACys; Group 3administered with 28AACysTTD (SEQ ID NO: 54); and Group 4 administeredwith 28AACysTTD (SEQ ID NO: 56).

(4) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(5) Blood Sampling

On Day 7 from the final 4th immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 7 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization Groups. As shown in Table 7, theantibody inducing ability was found in the groups administered with28AACys (SEQ ID NO: 6), 28AACysTTD (SEQ ID NO: 54) or 28AACysEIFEFTTD(SEQ ID NO: 56). Thus, it was found that the antibody inducing abilityof Aβ sequence with addition of Cys remained even if an additionalexogenous peptide sequence was bound to said Aβ sequence.

TABLE 7 Example 8 Nasal, intradermal and oral administration of Aβpeptide with addition of cysteine (1) Preparation of Aβpeptide with addition of cysteine 35AACys: (SEQ ID NO: 21)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMC

The above peptide was synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution, which wasstored at −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 9 mice were divided into 3 groups each comprising 3 mice: Group 1nasally administered; Group 2 intradermally administered; and Group 3orally administered.

(4) Immunization and Schedule

Primary Immunization

Common to each of Groups, to 200 μL of the stock solution was added 1800μL of saline to 0.5 mg/mL of the concentration and 200 μL of the mixturewas administered to mice using a 1 mL tuberculin syringe (Terumo,SS-01T2613S) intradermally or subcutaneously at the abdomen (dose permouse: 100 μg). The 2nd, 3rd and 4th (final) immunizations of the miceof each group were conducted at weekly intervals after the primaryimmunization as described below.

Group 1: Nasal Administration

Each 20 μL of the stock solution was administered to mice via the nasalcavity using Pipetman P-20 (Gilson) (dose per mouse: 100 μg).

Group 2: Intradermal Administration

On the day before the administration the back of mice was shaved. Underanesthetization with isoflurane, the shaved area was disinfected with70% alcohol and dried before administration and then 20 μL of the stocksolution was administered dropwise to mice using Pipetman P-20 (Gilson)(dose per mouse: 100 μg).

Group 3: Oral Administration

For the 2nd and 3rd immunizations, to 200 μL of the stock solution wasadded 2300 μL of saline to 0.2 mg/mL of the concentration and each 500μL/mouse of the mixture was administered into the stomach of mice usinga probe for oral use (Natsume Seisakusho CO LTD., KN-348, for mice)attached to a 1 mL tuberculin syringe (Terumo, SS-01T2613S) (dose permouse: 200 μg). For the 4th (final) immunization, to 100 μL of the stocksolution was added 2400 μL of saline to 0.2 mg/mL of the concentrationand each 500 μL/mouse of the mixture was administered into the stomachof mice using a probe for oral use attached to a 1 mL tuberculin syringe(dose per mouse: 100 μg).

(5) Blood Sampling

On Day 6 from the primary immunization, on Day 6 from the 2ndimmunization and on Day 6 from the 3rd immunization, 50 to 150 μL ofblood was collected from the tail vein. Further on Day 7 from the final4th immunization, blood was collected from the abdominal aorta of allmice under anesthesia with pentobarbital sodium (Kyoritsu SeiyakuCorporation, Somnopentyl). The sampling blood was transferred to theMicrotainer (Becton Dickinson Co., Ltd), adequate clotting has occurredat room temperature and then centrifuged at 5,000 rpm for 10 min. Eachof the separated serum was dispensed into two 0.5 mL tubes and stored at−80° C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 8 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization Groups. As shown in Table 8, it wasfound that Aβ peptide fragment with addition of cysteine showed theantibody inducing ability against Aβ irrespective of nasal, percutaneousor oral administration.

TABLE 8 Example 9 Comparison of antibody inducing ability of inintradermal administration between Aβ peptideswith and without addition of cysteine (1) Preparation of Aβpeptides with and without addition of cysteine 35AA: (SEQ ID NO: 20)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM 35AACys: (SEQ ID NO: 21)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMC

The above peptides were synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution, which wasstored at −80° C. or lower till use.

(2) Immunized Mice

Male C57BL/6 mice (7 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(3) Immunization Groups

The 10 mice were divided into 2 groups each comprising 5 mice: Group 1administered with 35 amino acids Aβ peptide with addition of cysteine;and Group 2 administered with 35 amino acids Aβ peptide without additionof cysteine.

(4) Immunization and Schedule

The primary immunization was conducted as in Example 8. The 2nd, 3rd and4th (final) immunizations were done at weekly intervals after theprimary immunization as described below. On the day before theadministration the back of mice was shaved. Under anesthetization withisoflurane, the shaved area was disinfected with 70% alcohol beforeadministration, application/peeling-off of a surgical tape (NichibanCo., Ltd.) was repeated 10 times to remove the corneal layer ofepidermis and then 20 μL of the stock solution was administered dropwiseto mice using Pipetman P-20 (Gilson) (dose per mouse: 100 μg).

(5) Blood Sampling

On Day 7 from the final 4th immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(6) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 9 shows the calculated anti-Aβ antibody titer of the murineserum in each of the immunization Groups. As shown in Table 9, it wasfound that Aβ peptide fragment with addition of cysteine showed a higherantibody inducing ability by 10-fold or more as compared to Aβ peptidewithout addition of cysteine.

TABLE 9 Example 10 Comparison of Aβ peptide with/without additionof cysteine between with and without addition of adjuvant(1) Preparation of Aβ peptides with/without addition of cysteine 28AA:DAEFRHDSGYEVHHQKLVFFAEDVGSNK (SEQ ID NO: 5) 28AACys:DAEFRHDSGYEVHHQKLVFFAEDVGSNKC (SEQ ID NO: 6)

The above peptides were synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution. To 100 μL ofthe stock solution was added 900 μL of saline to 0.5 mg/mL of theconcentration and the mixture was dispensed into 1.5 mL tube (immunogen)and stored at −80° C. or lower till use.

(2) Adjuvant

As an adjuvant, the commercially available Quil-A (Accurate Chemical &Scientific Corporation) and MPL+TDM emulsion (Corixa) were used. Quil-Awas dissolved in saline to obtain 5 mg/mL of a stock solution. Inaddition, 1 vial of MPL+TDM emulsion was dissolved in 1 mL of saline toobtain a stock solution, which was stored at 4° C. till use.

(3) Immunized Mice

Male C57BL/6 mice (9 weeks old, SPF) were purchased from Japan CharlesRiver Co., Ltd. and bred in SPF environment.

(4) Immunization Groups

The 24 mice were divided into 6 groups each comprising 4 mice: Group 1administered with 28AA; Group 2 administered with 28AA+Quil-A; Group 3administered with 28AA+MPL+TDM emulsion; Group 4 administered with28AACys; Group 5 administered with 28AACys+Quil-A; and Group 6administered with 28AACys+MPL+TDM emulsion.

(5) Dose and Preparation of Sample for Administration

A dose of Aβ peptide was 100 μg per animal and 0.2 mL of 500 μg/mL ofsample for administration was administered. With respect to the groupadministered with 28AA and the group administered with 28AACys, 20 μL ofeach of the stock solution and 180 μL of saline were mixed and themixture was stored at −80° C. until the administration. With respect tothe group administered with 28AA+Quil-A and the group administered with28AACys+Quil-A, 20 μL of each of the stock solution, 10 μL of 5 mg/mLthe stock solution of Quil-A (50 μg per animal) and 170 μL of salinewere mixed and the mixture was stored at −80° C. until theadministration. With respect to the group administered with 28AA+MPL+TDMemulsion and the group administered with 28AACys+MPL+TDM emulsion, 90 μLof each of the stock solution and 660 μL of saline were mixed toprepared the stock solution for administration, which was stored at −80°C. 0.5 mL of the MPL+TDM emulsion stock solution was added just beforeadministration to obtain suspension (for 4.5 individual).

(6) Immunization and Schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 3 timesat 2-week intervals.

(7) Blood Sampling

On Day 7 from the final 3rd immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(8) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove. Table 10 shows the calculated anti-Aβ antibody titer of themurine serum in each of the immunization Groups. As shown in Table 10,it was found that antibody induction against Aβ could hardly be observedfor 28AA together with Quil-A but observed for 28AA together withMPL+TDM emulsion. In case of 28AACys, a high antibody titer could beobserved either with Quil-A or with MPL+TDM emulsion. The antibodyinduction of 28AACys alone was similar to that of 28AA together withMPL+TDM emulsion.

TABLE 10 Example 11 Pharmacological evaluation of Aβ peptide withaddition of cysteine using Alzheimer disease model mice(1) Preparation of Aβ peptide with addition of cysteine 35AACys:(SEQ ID NO: 21) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMC

The above peptide was synthesized (Hokkaido System Science Co., Ltd.)and diluted with saline to obtain a 5 mg/mL stock solution. To 100 μL ofthe stock solution was added 900 μL of saline to 0.5 mg/mL of theconcentration and the mixture was dispensed into 1.5 mL tube (immunogen)and stored at −80° C. or lower till use.

(2) Immunized Mice

Transgenic mice (TG2576, female, 11 weeks old, SPF), which showedAlzheimer-like pathological condition with accumulation of human Aβ inthe brain through expression of a human Aβ precursor protein, werepurchased from Taconic Farms, Inc. and bred in SPF environment.

(3) Immunization Groups

The 6 mice were divided into 2 groups each comprising 3 mice: Group 1administered with 35AACys; and Group 2 not administered (control).

(4) Immunization and schedule

Each 200 μL/mouse of an immunogen was administered to mice using a 1 mLtuberculin syringe (Terumo, SS-01T2613S) intradermally or subcutaneouslyat the abdomen (dose per mouse: 100 μg). The mice were immunized 4 timesat 2-week intervals from 18-week old and then at monthly intervals witha total of 11 immunizations.

(5) Blood Sampling

On Day 13 from the final immunization, blood was collected from theabdominal aorta of all mice under anesthesia with pentobarbital sodium(Kyoritsu Seiyaku Corporation, Somnopentyl). The sampling blood wastransferred to the Microtainer (Becton Dickinson Co., Ltd), adequateclotting has occurred at room temperature and then centrifuged at 5,000rpm for 10 min. Each of the separated serum was dispensed into two 0.5mL tubes and stored at −80° C. or lower until measurement.

(6) Extraction of Human Aβ Deposited in Brain

After blood sampling, the cerebrum was isolated via craniotomy. A partof the frontal lobe was sectioned and weighed. Thereto was added TBScontaining a proteinase inhibitor (Roche, Complete Protease InhibitorCocktail Set, 1 tablet/50 mL of solution) (20 mM Tris, 137 mM NaCl,pH7.6; hereinafter referred to as “CP/TBS”) to 150 mg (wet weight ofbrain)/mL and the mixture was homogenized with a homogenizer made ofTeflon (trademark). Then, the mixture was centrifuged at 12,000 g at 4°C. for 10 min. The supernatant was discarded and the resultingprecipitate was resuspended in 1% TritonX-100/CP/TBS (in the same amountas that of CP/TBS above) and the suspension was vortexed for 1 min. Thesuspension was centrifuged at 12,000 g at 4° C. for 10 min. Thesupernatant was discarded and the resulting precipitate was resuspendedin 2% SDS/CP/TBS (in the same amount as that of CP/TBS above) and thesuspension was vortexed for 1 min. The suspension was furthercentrifuged at 12,000 g at 4° C. for 10 min. The resulting supernatantwas used as a sample fraction containing the human Aβ deposited in thebrain.

(7) Measurement of Anti-Aβ IgG Antibody

Measurement of the anti-Aβ IgG antibody was conducted as describedabove.

(8) Measurement of Human Aβ Deposited in Brain

Measurement was performed using the human β amyloid (1-42) ELISA kitWAKO (WAKO, code number 296-64401). To a microtiter plate immobilizedwith a human antibody against Aβ (BAN50) was added a sample containing50-fold diluted human Aβ at 100 μL/well. After overnight reaction at 4°C., each of the diluted samples added was discarded and the microtiterplate was washed 5 times with 350 μL/well of the wash solution in thekit. An HRP labeled antibody (BC05) solution (100 μL) was added to eachof the wells followed by 1 hour reaction at 4° C. After the reaction,the labeled antibody solution was discarded and the microtiter plate waswashed 5 times with 350 μL/well of the wash solution. A TMB solution(chromogenic agent; 100 μL) was added to each of the wells, followed by30 min. reaction at room temperature in the dark. Then, 100 μL/well ofthe stop solution was added to quench the enzymatic reaction and opticaldensity at 450 nm was measured (OD450 value). A standard curve was madeusing the attached standard solution (human β amyloid (1-42), 20pmoL/L). On the measurement of the samples, said standard solution wasdiluted with the standard dilution solution to 0.156, 0.3125, 0.625,1.25, 2.5, 5, 10 pmol/mL. OD450 value of each of the diluted standardsolution was measured simultaneously with the measurement of each of thesample. A human Aβ concentration in each of the samples was calculatedusing the resulting unit of the standards and the standard curve ofOD450 value.

Tables 11 and 12 show the calculated anti-Aβ antibody titer in themurine serum and the concentration of human Aβ deposited in the brain ofeach of the immunization groups. As shown in Table 11, production ofantibody against Aβ was observed for Alzheimer model mice immunized with35AACys. Besides, as shown in Table 12, a concentration of human Aβ(hAβ) deposition in the brain was found to be lower than that withAlzheimer model mice with no administration. Thus, it was proved that Aβpeptide with addition of Cys was efficacious not only as a prophylactic(peptide vaccine) but also as a therapeutic agent.

-   Table 11

Antibody titer (ng/mL) Animal No. Group 1 2 3 Mean Group administered43085 159564 308485 170378.0 with 35AACys Group not administered 242 105660 335.2

-   Table 12

hA β conc. (pmol/mL) Animal No. Group 1 2 3 Mean Group administered 5441137 2377 1353.0 with 35AACys Group not administered 4170 6379 44424997.1

INDUSTRIAL APPLICABILITY

The immunogenic peptide that induces an enhanced immune responsecomprising an Aβ peptide or a portion thereof with addition or insertionof cysteine or with addition of a peptide containing cysteine and thegene fragment encoding said peptide of the present invention may be usedas a safe and convenient means for immune stimulation in a peptidevaccine, a DNA vaccine and the like. Moreover, the Aβ peptide thatinduces an enhanced immune response or a portion thereof, prepared by amethod for enhancing an immune response of the present invention, may bean efficacious medicament for preventing or treating of Alzheimerdisease.

The invention claimed is:
 1. An immunogenic peptide that induces anenhanced immune response consisting of amino acids selected from thegroup consisting of the 1st-29th, 1st-30th, 1st-31st, 1st-35th,1st-36th, 1st-37th, 1st-38th, 1st-39th, 1st-41st and 1st-42nd of anamyloid β peptide with the insertion of cysteine-between the 18th and19th amino acid residues, between the 25th and 26th amino acid residues,or between the 28th and 29th amino acid residues counted from theN-terminus of the amyloid β peptide.
 2. The immunogenic peptideaccording to claim 1, wherein cysteine is inserted into the amyloid βpeptide between the 18th and 19th amino acid residues counted from theN-terminus of the amyloid β peptide.
 3. The immunogenic peptideaccording to claim 1, wherein cysteine is inserted into the amyloid βpeptide between the 25th and 26th amino acid residues counted from theN-terminus of the amyloid β peptide.
 4. The immunogenic peptideaccording to claim 1, wherein cysteine is inserted into the amyloid βpeptide between the 28th and 29th amino acid residues counted from theN-terminus of the amyloid β peptide.
 5. The immunogenic peptideaccording to claim 1, wherein the amyloid β peptide consists of theamino acid sequence of SEQ ID NO:
 34. 6. A medicament comprising theimmunogenic peptide according to claim 1 as an active ingredient, and apharmaceutically acceptable excipient.
 7. A medicament comprising theimmunogenic peptide according to claim 1 as an active ingredient, apharmaceutically acceptable excipient, and an adjuvant.
 8. A medicamentcomprising the immunogenic peptide according to claim 1 as an activeingredient, and a pharmaceutically acceptable excipient, with theproviso that the medicament does not comprise an adjuvant.